It has long been recognized that when oxygenation is restored in hypoxic tissues (re-oxygenation), hypoxic tissue injury is further augmented, a process called ischemia-reperfusion injury (MI). This mechanism of cellular injury is especially prominent in surgical, myocardial, hepatic, intestinal, cerebral, renal, and other ischemic syndromes and occurs, with varying degrees of severity, after all forms of organ transplantation. Vascular oxidative stress is also a major cause of pulmonary and systemic pathology in conditions including Acute Lung Injury (ALI/ARDS), inflammatory lung conditions, sepsis, hyperoxia and radiation injury.
Vascular I/R is responsible for acute graft failure and delayed complications of organ transplantation and cardiopulmonary bypass, as well as necrotic and apoptotic tissue injury in acute myocardial infarction (AMI), stroke, thrombosis and other cases of occlusive ischemia of vessels that perfuse blood to internal organs or extremities.
Oxidative stress in general, is characterized by the formation of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and hydroxyl radical. These molecules are highly reactive and react with structures such as DNA, key cellular proteins, and the lipid component of the cell membrane leading to lipid peroxidation and subsequent cell injury that can be detected by increased permeability, and in more severe cases to cell lysis. The generation of intracellular ROS occurs in most lung parenchymal cells, such as endothelial cells, Type II alveolar epithelial cells, Clara cells, alveolar epithelial cells as well as in alveolar macrophages (See FIG. 1—taken from Muzykantov, V. R. 2001. Targeting of superoxide dismutase and catalase to vascular endothelium. J Controlled Release 71:1-21).
There are at least two important mechanisms for ROS production during IRI. During anoxia, hypoxanthine accumulates and the enzyme xanthine dehydrogenase is converted into xanthine oxidase, This is followed by the degradation of hypoxanthine into superoxide which occurs during reoxygenation. The other mechanism depends on the NADPH oxidase system, which is present mainly on the membrane surface of neutrophils and monocytes/macrophages and endothelial cells and catalyzes the reduction of oxygen into hydrogen peroxide and superoxide anion. Classically, it has been thought that such activated neutrophils contribute to vascular reperfusion injury, although cellular injury is propagated in the absence of inflammatory cells through mechanisms involving reactive oxygen (ROS) or nitrogen species (RNS).
In addition to leukocyte ROS, generation of ROS throby endothelial cells through this pathway also appears to be important. Studies from our group and others indicate that vascular oxidative stress induced by ROS, (including superoxide anion and H2O2) plays a key role in ischemia-reperfusion (I/R) injury. Endothelial cells (EC), which line the luminal surface of blood vessels and control vascular tone, transport of blood components to tissues and maintain blood fluidity) represent a main target of ROS in I/R. EC dysfunction and damage induced by ROS thus play a key role in the initiation and propagation of I/R injury (see FIG. 1).
Ironically, intracellular ROS produced by EC themselves in response to ischemia (endogenous ROS production) help to initiate the injurious I/R cascade, while extracellular ROS (exogenous ROS production) released from activated white blood cells (WBC) augment and further propagate the vicious cycle and subsequent pathological reactions including WBC adherence, thrombosis, vascular edema and vasoconstriction, i.e., events initiated by EC damage.
Given this pathophysiology, many believe that I/R injury could be ameliorated or even prevented by effective ROS detoxification, thus justifying the interest in the development of antioxidant prophylaxis and therapies. Conventional antioxidants such as N-acetyl-cysteine (a precursor for a main cellular reducing agent, glutathione), selected vitamins (e.g., tocopherol) and food supplements (flavonoids) afford some degree of protection in selected cases of modest chronic oxidative stress. However, the potency of these antioxidants has not been sufficient to protect against severe acute and sub-acute forms of vascular oxidative stress, such as I/R. More effective approaches are therefore needed.
Correspondingly, lung transplantation has become an important therapy for many end-stage lung diseases. Unfortunately, due to the circulatory disruption required by transplantation, a significant cause of early morbidity and mortality associated with this procedure is ischemia-reperfusion-induced injury (IRI) of the lung. Oxidative stress, the key mediator of IRI, typically manifests itself within the first 72 hours after transplantation and is characterized by alveolar damage, lung edema, and hypoxemia. Despite advances in our understanding of the mechanisms of IRI, and improvements in the technique of lung preservation, in surgical techniques and in perioperative care, up to 15% of all transplanted lungs will end up with primary graft failure. Better ways to deliver potent and safe antioxidant agents are clearly needed.
The usefulness of thoracic radiotherapy is greatly limited by the sensitivity of the lung tissue to irradiation doses necessary to eradicate malignant cells. Clinically significant radiation lung injury, such as pneumonia-like inflammation and late stage fibrosis, occurs in up to 30% of patients irradiated for lung cancer and about 10-15% of other thoracic oncology patients. The need, however, to protect “normal” lung parenchyma from unacceptable radiation injury compromises the ability to deliver tumoricidal radiotherapy doses and contributes to the high local recurrence rates experienced by lung cancer patients following definitive radiotherapy. The cytotoxic effects of ionizing radiation in normal lung parenchyma are mediated by the generation of reactive oxygen species (ROS) and propagated by ROS-driven oxidative stress thus identifying a central role of tissue antioxidant defense. A safe radioprotecting agent that would ameliorate radiation toxicity while not protecting tumor, or even preferably radiosensitizing tumor cells is desperately needed. We and others have shown that antioxidant enzyme therapy alleviates radiation-induced fibrotic lung disease. NF-E2-related factor 2 (Nrf2), a key transcriptional regulator for antioxidant response element (ARE) mediates induction of cellular antioxidant and detoxifying enzymes. Preliminary data obtained from an exploratory R21 award showed that whole grain dietary flaxseed (FS) boosts Nrf2-mediated antioxidant defense in murine lungs. Importantly, dietary whole-grain FS ameliorated the adverse effects of thoracic radiation by enhancing survival and blocking lung fibrosis while, remarkably inhibiting lung tumor growth and metastasis. We have evidence to believe that the bioactive ingredient(s) in the FS grain that mediate these effects are the lignans. Flaxseed contains the lignan precursor secoisolariciresinol diglucoside (SDG) which is metabolized in the intestine to mammalian lignans which are safe, compounds with known antioxidant, anti-inflammatory and anticarcinogenic effects. Our group discovered that chemically synthesized, commercially available flaxseed lignans, activate the Nrf2/ARE pathway mediating transcription of antioxidant enzyme genes and inhibit lung cancer cell proliferation in vitro. Additional evidence revealed proteasomal inhibition as a potential mechanism of their action. We, therefore, believe that coordinate induction of Nrf2/ARE regulated antioxidant genes by flaxseed lignans may be a novel therapeutic strategy to alleviate radiation pneumonopathy and that these agents are responsible for inhibition of lung tumor growth and metastasis.